R. Vishnubhotla, S. Robinson, C. Montgomery, J. Askim, S. Semancik
National Institute of Standards and Technology,
Keywords: biosensor, aptamer, DNA, electrochemistry
Summary:Biosensing devices will become increasingly important in areas such as pharmaceutical discovery, biomanufacturing and healthcare screening. Nucleic acids are promising probes for the detection of markers such as other nucleic acids, proteins, and even volatile organic compounds. Aptamers, or oligonucleotide sequences selected to bind to particular targets, are growing in significance for biomarker detection. The conformation of immobilized DNA structures is critical for sensing applications since it affects binding events and biomolecular stability. However, conformational effects in varied environmental conditions are not always well understood. In the work described here, we have used temperature-controlled electrochemical microdevices  (Fig. 1) for small-volume studies of DNA conformation, binding events and stability changes  of immobilized polythymine (polyT), streptavidin aptamer (StreapApt2) and PDGF aptamer. To define the interfacial systems being studied, we employed surface characterization techniques such as x-ray photoemission spectroscopy (XPS) and atomic force microscopy (AFM) to characterize surface composition and morphology. PolyT with a length of 20 nucleotides and a methylene blue (MB) redox tag at the 3’ end was used as a simple model in our initial work to correlate electrochemical current measurements with conformation changes. The MB tag exchanges electrons with the Au working electrode of the device, and this current was measured as a function of temperature. Various stressors such as temperature changes, pH variation, and salt concentration of a phosphate buffer saline (PBS) working buffer were examined. The polyT strand demonstrates a high current for low temperature, which drops off as temperature increases. While this behavior occurs regardless of the PBS pH, different profiles are observed as the salt concentration of the PBS is changed. We have also studied the behavior of a polythymine strand with an additional anthraquinone (AQ) reference tag at the 5’ end to better define the conformation of the full DNA strand. Preliminary studies on aptamers were also completed, specifically with a streptavidin aptamer (StrepApt2) and a platelet derived growth factor (PDGF) aptamer. The current versus temperature signals of streptavidin aptamer with and without a bound streptavidin protein are shown in Fig.2. Further studies include the binding of PDGF to its aptamer and determining the effects of persistence length and loss of rigidity on signal strength by varying the length of the polyT strand. References:  Shen, Z., Sintim, H.O., Semancik, S. Rapid Nucleic Acid Melting Analyses Using a Microfabricated Electrochemical Platform. Anal. Chim. Acta, (2015), 853, 265-270  Robinson, S.M.; Shen, Z.; Askim, J.R.; Montgomery, C.B.; Sintim, H.O.; Semancik, S. Ligand-Based Stability Changes in Duplex DNA Measured with a Microscale Electrochemical Platform. Biosensors (2019), 9, 54.